Rapid remodeling of the microtubule cytoskeleton is essential for normal cell division, motility and morphogenesis. A unique and intriguing class of proteins involved in this remodeling are the microtubule severing enzymes, so named because of their ability to generate internal breaks in the microtubule lattice, in vitro. The studies outlned in this research proposal will elucidate the cellular functions and biophysical mechanisms of action of members of a still poorly understood subfamily of microtubule severing enzymes, termed fidgetins. The founding member of this subfamily, fidgetin, has long been known to be important for mammalian development, yet the mechanistic basis of its developmental functions remains unclear. In a recent study, we showed that human fidgetin is a microtubule severing enzyme and minus-end depolymerase. We have now found that fidgetin and the closely related protein fidgetin-like 2 perform fundamental but distinct roles in the regulation of human cell migration. Fidgetin localizes to the centrosome and normally promotes cell motility. Cells depleted of fidgetin display severe reduction in motility rates. In stark contrast, fidgetin-like 2 associates with microtubules at the cell edge and normally functions to suppress cell movement. Cells lacking fidgetin-like 2 display a several fold increase in their rate of movement. Moreover, we have found that depletion of fidgetin-like 2 promotes wound healing and neovascularization in animal models. We will pursue the following two specific aims that, together, test the central hypothesis that fidgetin and fidgetin-like 2 recognize and modify distinct microtubule subpopulations thereby controlling different parameters of cell movement: Aim 1: Test the hypothesis that Fidgetin normally promotes cell motility by selectively severing and releasing microtubule minus-ends from centrosomes. Aim 2: Test the hypothesis that Fidgetin-like 2 normally suppresses cell motility by shearing the plus-ends of dynamic microtubules positioned at the cell edge. Our research plan combines complementary state-of-the-art biophysical and cellular approaches to systematically determine 1) how fidgetin and fidgetin-like 2 catalyze the removal of tubulin from the microtubule lattice, 2) how these activities are harnessed in cells to model MT arrays, and 3) how the modeling of cellular MTs by fidgetin or fidgetin-like 2 is translated into altered cell motility. Work will be carried out under the co-direction of David Sharp, an expert cell and molecular biologist, and Jennifer Ross, an expert single molecule biophysicist. Successful completion of the proposed work will provide fundamental insights into the basic mechanisms of microtubule regulation of cell motility, which is a central process in human development and health, and also foundationally establish a body of knowledge for the potential development of novel therapeutic paradigms to enhance tissue regeneration and repair through the manipulation of cell movement.